It is known that various factors may be responsible for limiting the growth of cells at high cell densities. These factors include absence of sufficient amounts of nutrients needed by the cells for sustained growth, as well as the presence of growth-limiting concentrations of inhibitors that may be secreted by the cells in culture. One inhibitor that is secreted by mammalian cells is ammonia. See Miller et at., Bioprocess Engineering, 3:113-122 (1988); Inlow et at., U.S. Pat. No. 5,156,964 describes a method for generating tolerance to ammonia that involves culturing cells in a medium to which ammonia has been added. Similarly, Schumpp et at., Cytotechnology, 8:39-44 (1992) describe a method for generating cell lines tolerant of both ammonia and lactic acid by culturing cells in a medium to which both ammonia and lactic acid had been added.
The previous methods have several drawbacks. First, in order to generate tolerance to an inhibitor according to the above methods, it is first necessary to determine that a particular inhibitor is a growth-limiting factor for cells and then to develop a protocol for generating tolerance to that inhibitor. Second, the growth of cell lines which are generated with tolerance to a particular inhibitor according to the above methods may then be limited by a second, different inhibitor. Repeated experiments may be necessary to generate tolerance to multiple growth-limiting inhibitors in order to achieve significant increases in cell densities.